1. Field of the invention
This invention relates to an aeration apparatus for the culture of mammalian cells. More particularly, it relates to an aeration apparatus that supplies the aeration that is necessary and sufficient for the growth of the cells when large-scale culture of mammalian cells is being done, so that the said cells can be cultured under appropriate environmental conditions.
2. Description of the prior art
Conventional culture of mammalian cells is done, for example, in the culture apparatus 1 shown in FIG. 2. This culture apparatus 1 has a culture vessel 6 with impeller 3. The culture vessel 6 is provided with an air supply inlet 4 and an exhaust outlet 5. When mammalian cells are to be cultured with the use of this apparatus, culture broth 2 that contains mammalian cells is placed inside the culture vessel 6, and air, air enriched with oxygen, or very pure oxygen (below, all referred to as `air`) is supplied, while the impeller 3 causes agitation. The air supplied is taken in through the surface of the culture broth 2 that is being agitated, supplying air to the mammalian cells that are in the culture broth 2.
With such an apparatus, when the volume of the culture vessel 6 is relatively small (about 100-1000 ml), the supply of air is adequate, but when the volume of culture vessel 6 is large (for example, 500-1000 liters), sufficient air is not supplied to the culture cells. That is, when culture is on an industrial scale, the ratio of the surface area to the volume of fluid in culture vessel 6 becomes small, and an aeration system by means of the diffusion of air does not provide the necessary and sufficient amount of air for the growth of the cells.
A number of means have been used for the supply of the necessary and sufficient amount of air for the growth of mammalian cells being cultured on an industrial scale (that is, for the purpose of increasing the volumetric oxygen-transfer coefficient.
The method for aeration of culture vessels on an industrial scale that is most widely used today is a direct method for air supply by means of bubble aeration and agitation with an air-blowing nozzle introduced into the culture broth. In this method, the tip of the hole in the air-blowing nozzle is provided with an orifice or a sintered filter or the like, and the diameter of the bubbles of air is made as small as possible, so that the general capacity coefficient for oxygen in the culture broth becomes as large as possible.
In the method for air supply by means of bubble aeration and agitation, the bubbling of the culture broth becomes pronounced. The reason is that when mammalian cells are to be cultured, there are proteins such as serum, etc., in the culture broth, and the said proteins act as surface-active agents. The bubbling of the culture broth inhibits the growth of the cells and decreases the production of product produced by the cells, when the vessel is filled with many bubbles.
In another method for the effective supply of air to culture cells, the partial pressure of oxygen can be increased within the upper limit above which cytotoxicity would occur, causing an increase in the volumetric oxygen-transfer rate. However, it is relatively difficult to control the partial pressure of oxygen, and results are not as good as might be expected.
As yet another method, which does not involve the direct supply of air to the culture cells as do the methods described above, there is a method in which air is supplied indirectly with the use of a semi-permeable membrane or a silicone membrane. By this method, on one side of the membranes mentioned above, air or a liquid medium that contains air is caused to flow, and air is supplied through the membrane to the culture cells on the other side. For example, a method is disclosed in Japanese Laid-Open Patent Application No. 60-234580 in which oxygen is supplied to the culture broth via a semi-permeable membrane. However, in this method, the resistance of the membranes mentioned above is great when air is permeating through them. For that reason, in order to assure the amount of air needed for the growth of the cells, it is necessary for the surface area of the membranes to be extremely large, and a large culture vessel is needed. Moreover, the disposition of the membranes in the culture vessel becomes very complicated. In particular, when the culture involves the use of a microcarrier, the microcarrier that is carrying the cells comes to be distributed in a very uneven way throughout the culture vessel, so that the cells in the areas that are relatively undisturbed die. The structure of the culture vessel is complex, which has the drawback of being expensive. Therefore, the indirect aeration method that involves the use of a membrane is not appropriate for use in large culture vessels on the industrial scale.
As mentioned above, there have not been any effective means for the aeration of mammalian cells being cultured in a large-scale culture apparatus until now.